Although the pubertal mammary gland of rodents is particularly sensitive to initiation of transformation, an absolute requirement for tumor development is post-initiation, post-pubertal exposure to ovarian hormones. This requirement for post-pubertal exposure to ovarian hormones is consistent with known breast cancer risk factors: early onset of menarche, and late age at menopause. Substantial evidence indicates that progesterone (P) has major mitogenic activity in the breast at sexual maturity and the increased risk of breast cancer associated with post-menopausal combined estrogen (E)+P hormone replacement therapy (HRT) indicates a role of P in the etiology of breast cancer. We hypothesize that increased lifetime exposure to P in combination with E underlies the increased risk of breast cancer associated with early onset of menarche, late age at menopause and E+P HRT. Earlier onset of menarche is predicted to result in earlier breast maturation and earlier exposure to the proliferative effects of P. Exposure to environmental stressors that cause earlier onset of menarche, mammary gland maturation and exposure to P are also predicted to increase breast cancer risk. In Collaborative Research Project I, we will test these hypotheses using the mouse mammary gland model. Our first goal is to address important gaps in our knowledge about the mechanisms of P action in normal mammary gland. Based upon preliminary immunohistochemical evidence we hypothesize that the transition from the pubertal to adult state of mammary gland development is due to differential regulation and expression of the progesterone receptor (PR) isoforms, PRA and PRB. We will 1) determine the topographic, cellular and hormonal regulation of PRA and PRB protein and mRNA expression in the pubertal vs. adult mammary gland. 2) Determine the functional roles of PRA and/or PRB and their interactions with growth factors in epithelial cell proliferation and examine PRA and PRB regulation of gene expression using cDNA array analysis.3) Determine the molecular mechanisms of differential PRA and PRB gene expression in the pubertal vs. adult mammary gland. Secondly, we will determine the effect of developmental exposure to environmental stressors that can affect pubertal development: estrogens(genestein), antiestrogens (ICZ, dioxin), and high fat diet (elevated insulin and IGF-I levels). Finally, we will determine the effect of perturbations in mammary gland pubertal development caused by these environmental stressors on subsequent susceptibility to tumor induction by a known mammary carcinogen N-methyl-nitrosourea (NMU).